PROJECT SUMMARY/ABSTRACT A class of cis-regulatory elements, called enhancers, play a central role in orchestrating spatiotemporally precise gene expression programs during development. Perturbations in enhancer sequence or regulation can lead to disease, including congenital malformations and cancer. Furthermore, enhancer sequence divergence is emerging as an important mediator of human phenotypic variation. A key feature of enhancers is their ability to activate transcription over long genomic distances of tens or even hundreds of kilobases away from their target promoters. Discovery that, when active, enhancers are marked by unique chromatin signatures, combined with genomic approaches such as ChIP-seq or Chromosomal Conformation Capture technologies (3C and derivatives) facilitated enhancer annotation across cell types and species and provided key insights into long-range regulation. Generally, however, in these population-level, fixed-cell assays, kinetic information underlying enhancer activation at a single-cell level has been lost. We recently developed a new imaging approach that allows us to label and track individual enhancer and promoter elements in living cells, in their native chromosomal context and in different cellular and activity states. Our proposed work further couples this technology with live-cell visualization of nascent transcripts to capture the kinetic behavior of enhancers and promoters and its relationship with the discontinuous nature of transcription. Using undifferentiated or differentiating stem cells as a cellular model, we will address major open questions in enhancer biology, including the real-time frequency and dynamics of enhancer-promoter contacts, their association with transcriptional bursts, and the role of chromatin topological organization in enhancer function. We plan to introduce a series of perturbations to investigate how disruption of specific events at enhancers, such as histone modification, variant incorporation or nucleosome remodeling, affects dynamics of long-range chromosomal contacts and transcriptional activation at the single-cell level. In complementary studies outlined in the second theme of the proposal, we are employing a diverse set of genomic and genetic approaches to identify novel factors that are required for long-range gene regulation and to define necessities and sufficiencies for enhancer activation within the native chromatin context. The two main themes will allow us to revisit current models of enhancer function (e.g. enhancer looping, enhancer delimitation by topologically associated domains, etc.) and will yield new concepts and mechanistic models of long-range gene control in mammals, with broad future implications for understanding and treatment of human disease.